Nonhuman animal model non-responsive to mycoplasma-origin lipoprotein/lipopeptide

ABSTRACT

An object of the present invention is to provide a non-human animal model unresponsive to a mycoplasma-derived lipoprotein/lipopeptide, and a method for screening an inhibitor or a promoter for a response to a mycoplasma-derived lipoprotein with the use of the non-human animal model. A non-human animal model whose function of a gene that encodes a protein such as TLR6 that specifically recognizes a mycoplasma-derived lipoprotein is deficient on its chromosome, for example, a TLR6 knockout mouse, is generated. With the use of the non-human animal model unresponsive to a mycoplasma-derived lipoprotein or an immune cell such as a macrophage derived from the non-human animal model, a subject material and a mycoplasma-derived lipoprotein, a response to a mycoplasma-derived lipoprotein in the non-human animal model or the immune cell is measured/evaluated, and then an inhibitor or a promoter for a response to that is screened.

TECHNICAL FIELD

The present invention relates to a non-human animal model unresponsiveto a mycoplasma-derived lipoprotein/lipopeptide, whose function of agene that encodes a protein such as TLR6 that specifically recognizes amycoplasma-derived lipoprotein/lipopeptide is deficient on itschromosome, and a method for screening an inhibitor or a promoter for aresponse to a mycoplasma-derived lipoprotein/lipopeptide with the use ofthe non-human animal model, etc.

BACKGROUND ART

It has been known that the Toll gene is required to control dorsoventralpatterning during the embryonic development of Drosophila (Cell 52,269-279, 1988; Annu. Rev. Cell Dev. Biol. 12, 393-416, 1996), and forantifungal immune responses in adult fly (Cell 86, 973-983, 1996). Ithas been clarified that the Toll is a type I transmembrane receptor withan extracellular domain containing leucine-rich repeat (LRR) and thatits cytoplasmic domain shows high homology to that of a mammalianinterleukin-1 receptor (IL-1R) (Nature 351, 355-356, 1991; Annu. Rev.Cell Dev. Biol. 12, 393-416, 1996; J. Leukoc. Biol. 63, 650-657, 1998).

Recently, mammalian homologs of Toll, designated as Toll-like receptors(TLRs), have been identified, and so far, six families including TLR2and TLR4 have been reported (Nature 388, 394-397, 1997; Proc. Natl.Acad. Sci. USA 95, 588-593, 1998; Blood 91, 4020-4027, 1998; Gene 231,59-65, 1999). It has been known that the TLR families, as in the case ofthe IL-1R mentioned above, recruit IL-1R-associated kinase (IRAK)through the adapter protein MyD88 and activate TRAF6, and then activateNF-êB in the downstream (J. Exp. Med. 187, 2097-2101, 1998; Mol. Cell 2,253-258, 1998; Immunity 11, 115-122, 1999). Further, the role of the TLRfamilies in mammals is also believed to participate in congenital immunerecognition as pattern recognition receptors (PRRs), which recognizebacterial cell common structures (Cell 91, 295-298, 1997).

It has been reported that one of such pathogen-associated molecularpatterns (PAMPs) to be recognized by the PRRs is a lipopolysaccharide(LPS), a major component of the outer membrane of Gram negative bacteria(Cell 91, 295-298, 1997), that the LPS stimulates host cells and makesthem produce various proinflammatory cytokines including TNFá, IL-1, andIL-6 (Adv. Immunol. 28, 293-450, 1979; Annu. Rev. Immunol. 13, 437-457,1995), and that the LPS captured by LPS-binding protein (LBP) isdelivered to CD14 on the cell surface (Science 249, 1431-1433, 1990;Annu. Rev. Immunol. 13, 437-457, 1995). The inventors of the presentinvention have constructed TLR4 knockout and TLR2 knockout mice, andhave reported that the TLR4 knockout mouse is unresponsive to LPS, amajor component of the outer membrane of Gram negative bacteriamentioned above (J. Immunol. 162, 3749-3752, 1999) and that a macrophageof the TLR2 knockout mouse decreased the reactivity of the TLR2 knockoutmouse to the cell wall of Gram positive bacteria and peptidoglycan, acomponent of the cell wall (Immunity, 11, 443-451, 1999).

On the other hand, Mycoplasma is the smallest microorganism that canself-propagate, and is biologically classified into bacteria. However,unlike other bacteria, Mycoplasma does not have a cell wall, andtherefore, it shows polymorphology and is unresponsive to cell wallsynthesis inhibitors such as penicillin and cephem. Though there areseven kinds of Mycoplasma which are often separated from human, onlyMycoplasma pneumoniae shows apparent pathogenicity and is known to causerespiratory infections such as upper respiratory infection, bronchitisand pneumonia. Recently, the present inventors have revealed that abacterial cell component such as a mycoplasma-derivedlipoprotein/lipopeptide causes vital reaction via TLR2 and MyD88signaling pathways (J. Immunol. 164, 554-557, 2000). However, a proteinthat specifically recognizes a mycoplasma-derivedlipoprotein/lipopeptide has been unknown, and consequently, themolecular mechanism that a mycoplasma-derived lipoprotein/lipopeptideactivates immune cells has not been elucidated sufficiently.

Though in vivo responses to bacterial cell components are expected tovary depending on the difference of expression levels of each TLR on thecell surface, the contribution of individual members of the TLR familyto signaling by bacterial cell components' stimuli in vivo remains to beelucidated. In addition, though it is known that a water-insolublelipoprotein/lipopeptide that is present on a biomembrane etc. activatesimmune cells, a protein that specifically recognizes amycoplasma-derived lipoprotein/lipopeptide has been unknown. An objectof the present invention is to provide a non-human animal modelunresponsive to a mycoplasma-derived lipoprotein/lipopeptide, whosefunction of a gene that encodes a protein that specifically recognizes amycoplasma-derived lipoprotein/lipopeptide is deficient on itschromosome, particularly a non-human animal whose function of the TLR6gene is deficient on its chromosome, which is useful for elucidating thecontribution of individual members of the TLR family to signaling bystimulation with a mycoplasma-derived lipoprotein/lipopeptide in vivo,especially the role of TLR6 in vivo, and a method for screening aninhibitor or a promoter for a response to a mycoplasma-derivedlipoprotein/lipopeptide with the use of the non-human animal model.

As aforementioned, as to TLR family in mammals, which is involved incongenital immune recognition as a pattern recognition receptor whichrecognizes bacterial cell common structures, six members of them(TLR1-6) have been reported (Nature 388, 394-397, 1997; Proc. Natl.Acad. Sci. USA, 95, 588-593, 1998; Gene 231, 59-65, 1999). However, areceptor that specifically recognizes a mycoplasma-derivedlipoprotein/lipopeptide has been unknown. The present inventors havegenerated TLR6 konckout mice as follows: cDNA of TLR6 which had beenidentified was isolated from mouse gene library; a genetic sitecontaining an intracellular domain and a transmembrane domain of theTLR6 gene was replaced with a neomycin-resistant gene, and a HSV-tk genewas introduced into each C-terminal side respectively, and ES cellclones doubly resistant to G418 and ganciclovir were screened; the EScell clones were injected into blastocysts of C57BL/6 mice; TLR6knockout mice whose function of TLR6 genes is deficient on theirchromosomes were born through the germline at the expected Mendelianratios. Subsequently the present inventors have found that TLR6 is areceptor protein that specifically recognizes a mycoplasma-derivedlipoprotein/lipopeptide by comparing/analyzing the TLR6 knockout mice,wild-type mice and TLR2 knockout mice, and the present invention hascompleted.

DISCLOSURE OF THE INVENTION

The present invention relates to a non-human animal model unresponsiveto a mycoplasma-derived lipoprotein/lipopeptide, whose function of agene that encodes a protein that specifically recognizes amycoplasma-derived lipoprotein/lipopeptide is deficient on itschromosome (claim 1), the non-human animal model unresponsive to amycoplasma-derived lipoprotein/lipopeptide according to claim 1, whereinthe protein that specifically recognizes a mycoplasma-derivedlipoprotein/lipopeptide is TLR6 (claim 2), the non-human animal modelunresponsive to a mycoplasma-derived lipoprotein/lipopeptide accordingto claim 1 or 2, wherein the non-human animal is a rodent (claim 3), thenon-human animal model unresponsive to a mycoplasma-derivedlipoprotein/lipopeptide according to claim 3, wherein the rodent is amouse (claim 4), the non-human animal model unresponsive to amycoplasma-derived lipoprotein/lipopeptide according to claim 4, whereinthe mouse is a TLR6 knockout mouse obtained by a process comprising thesteps of: a targeting vector is constructed by replacing the whole or apart of a gene fragment of a genetic site containing an intracellulardomain and a transmembrane domain of the TLR6 gene, which is obtained bya screening from a mouse gene library with the use of a probe derivedfrom a mouse EST clone, with a plasmid having a poly A signal and amarker gene; the targeting vector is linearized and then introduced intoan embryonic stem cell; the targeted embryonic stem cell deficient inTLR6 gene function is microinjected into a mouse blastocyst to constructa chimeric mouse; a heterozygous mouse was generated by mating thechimeric mouse and a wild-type mouse, and the heterozygous mice areintercrossed (claim 5).

The present invention also relates to a method for screening aninhibitor or a promoter for a response to a mycoplasma-derivedlipoprotein/lipopeptide wherein with the use of an immune cell derivedfrom the non-human animal model unresponsive to a mycoplasma-derivedlipoprotein/lipopeptide according to claims 1 to 5, a subject materialand a mycoplasma-derived lipoprotein/lipopeptide, a response to amycoplasma-derived lipoprotein/lipopeptide in the immune cell ismeasured/evaluated (claim 6), a method for screening an inhibitor or apromoter for a response to a mycoplasma-derived lipoprotein/lipopeptidewherein with the use of the non-human animal model unresponsive to amycoplasma-derived lipoprotein/lipopeptide according to claims 1 to 5, asubject material and a mycoplasma-derived lipoprotein/lipopeptide, aresponse to a mycoplasma-derived lipoprotein/lipopeptide in thenon-human animal is measured/evaluated (claim 7), the method forscreening an inhibitor or a promoter for a response to amycoplasma-derived lipoprotein/lipopeptide according to claim 6 or 7,wherein in the measurement/evaluation of the response to amycoplasma-derived lipoprotein/lipopeptide, the response is evaluated incomparison with a case using a wild-type non-human animal as a control(claim 8), the method for screening an inhibitor or a promoter for aresponse to a mycoplasma-derived lipoprotein/lipopeptide according toany one of claims 6 to 8, wherein the inhibitor or the promoter for aresponse to a mycoplasma-derived lipoprotein/lipopeptide is an inhibitoror a promoter for a mycoplasma infection (claim 9), the method forscreening an inhibitor or a promoter for a response to amycoplasma-derived lipoprotein/lipopeptide according to claim 9, whereinthe mycoplasma infection is human mycoplasma pneumonia, bovinepleuropneumonia, ovine/caprine mastitis or chicken respiratory disease(claim 10), the method for screening an inhibitor or a promoter for aresponse to a mycoplasma-derived lipoprotein/lipopeptide according toany one of claims 6 to 8, wherein the inhibitor or the promoter for aresponse to a mycoplasma-derived lipoprotein/lipopeptide is an agonistor an antagonist of TLR6 (claim 11).

The present invention further relates to an inhibitor or a promoter fora response to a mycoplasma-derived lipoprotein/lipopeptide obtained bythe method for screening an inhibitor or a promoter for a response to amycoplasma-derived lipoprotein/lipopeptide according to any one ofclaims 6 to 11 (claim 12), the inhibitor or the promoter for a responseto a mycoplasma-derived lipoprotein/lipopeptide according to claim 12,wherein the inhibitor or the promoter for a response to amycoplasma-derived lipoprotein/lipopeptide is an inhibitor or a promoterfor a mycoplasma infection (claim 13), the inhibitor or the promoter fora response to a mycoplasma-derived lipoprotein/lipopeptide according toclaim 13, wherein the mycoplasma infection is human mycoplasmapneumonia, bovine pleuropneumonia, ovine/caprine mastitis or chickenrespiratory disease (claim 14), the inhibitor or the promoter for aresponse to a mycoplasma-derived lipoprotein/lipopeptide according toclaim 12, wherein the inhibitor or the promoter for a response to amycoplasma-derived lipoprotein/lipopeptide is an agonist or anantagonist of TLR6 (claim 15).

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 is a graph showing gene maps of the TLR6 knockout mice and thewild-type mice of the present invention.

FIG. 2 is a graph showing the results of Southern blot analysis of theTLR6 knockout mice of the present invention.

FIG. 3 is a graph showing the results of TNFá production caused bystimulation with lipoprotein, LPS or PGN in the TLR6 knockout mice andthe wild-type mice of the present invention.

FIG. 4 is a graph showing the production of TNF-á or NO₂ ⁻ caused bystimulation with lipopeptide in the TLR6 knockout mice and the wild-typemice of the present invention.

FIG. 5 is a graph showing the results of NF-êB activation caused bystimulation with lipopeptide in the TLR6 knockout mice and the wild-typemice of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The protein that specifically recognizes a mycoplasma-derivedlipoprotein/lipopeptide according to the present invention is notparticularly limited as long as it is a protein that can specificallyrecognize a mycoplasma-derived lipoprotein/lipopeptide, and a part orthe whole of TLR6 is exemplified as a specific example. The protein thatspecifically recognizes a mycoplasma-derived lipoprotein/lipopeptide canbe prepared by known methods on the basis of its DNA sequenceinformation etc. Further, the mycoplasma-derived lipoprotein/lipopeptidein the present invention means, other than a lipoprotein/lipopeptidederived from Mycoplasma, Mycoplasma itself or a substance treated byMycoplasma, a lipopeptide derived from a synthetic Mycoplasma such asMALP-2, etc.

In the present invention, a non-human animal model unresponsive to amycoplasma-derived lipoprotein/lipopeptide means a non-human animalwherein a living organism, or cells, tissues or organs which comprise aliving organism show low or no reactivity specifically to thestimulation with a mycoplasma-derived lipoprotein/lipopeptide incomparison with wild-type non-human animals, that is, a non-human animalsuch as a mouse, a rat or a rabbit, wherein a living organism, or cells,tissues or organs which comprise a living organism show low or noreactivity specifically to the stimulation with a mycoplasma-derivedlipoprotein/lipopeptide, in spite that the living organism, or thecells, the tissues, or the organs which comprise the living organismshow normal reactivity to the stimulation with a lipoprotein/lipopeptidefrom Spirochaeta, Gram negative bacteria etc. As a specific example, anon-human animal whose function of a TLR6 gene is deficient on itschromosome, such as a TLR6 knockout mouse, is exemplified. Further, theabove-mentioned stimulation with a mycoplasma-derivedlipoprotein/lipopeptide includes in vivo stimulation wherein amycoplasma-derived lipoprotein/lipopeptide is administered to a livingorganism, and in vitro stimulation wherein a cell separated from aliving organism is made to contact with a mycoplasma-derivedlipoprotein/lipopeptide.

Next, a method for generating the non-human animal model unresponsive toa mycoplasma-derived lipoprotein/lipopeptide of the present invention isexplained with an example of a TLR6 knockout mouse. A gene that encodesTLR6 is screened with a gene fragment obtained from a mouse gene libraryby PCR or other such methods, and the screened gene that encodes theTLR6 is subcloned with a viral vector etc., then specified by DNAsequencing. The whole or a part of the gene that encodes TLR6 isreplaced with pMC1 neo gene cassette or the like, and a gene such as adiphtheria toxin A fragment (DT-A) gene or a herpes simplex virusthymidine kinase (HSV-tk) gene is introduced into 3′-terminal side toconstruct a targeting vector.

This constructed targeting vector is linearized, and introduced into EScells by a method such as electroporation, then the ES cells arehomologously recombined, and subsequently ES cells wherein homologousrecombination is caused by G418, ganciclovir (GANC) or other suchantibiotics are selected from the homologous recombinants. It ispreferable to confirm by Southern blotting etc. whether these selectedES cells are the object recombinants. A clone of the confirmed ES cellis microinjected into a blastocyst of a mouse, and the blastocyst istransplanted into a uterus of a recipient mouse to generate a chimericmouse. A heterozygous mouse (F1 mouse: +/−) can be obtained byintercrossing the chimeric mouse with a wild-type mice, and a TLR6knockout mouse of the present invention can be generated byintercrossing the heterozygous mice. In addition, as a method forconfirming whether TLR6 is present in the TLR6 knockout mouse, forinstance, there are Northern blotting or other such methods with whichRNA isolated from the mouse obtained by the above-mentioned method isexamined, and Western blotting or other such methods with which theexpression of TLR6 in the mouse is examined.

It is possible to confirm that the generated TLR6 knockout mouse isunresponsive to a mycoplasma-derived lipoprotein/lipopeptide, forexample, by contacting a mycoplasma-derived lipoprotein/lipopeptide withan immune cell such as a macrophage, a monocyte or a dendric cell of theTLR6 knockout mouse in vitro or in vivo, and then measuring theproduction amounts of TNF-á, IL-6, IL-12, IFN-ã etc. in the cells, theproliferative responses of splenic B cells, the expression amounts ofantigens such as CD40, CD80, CD86 and MHC class II on the surface ofsplenic B cells, and the activation of molecules in signaling pathwaysof TLR6, such as NF-êB, JNK and IRAK. Further, the TLR6 knockout mouseof the present invention can be used as a useful model for elucidatingan action mechanism of a mycoplasma-derived lipoprotein/lipopeptide andfor projecting a therapeutic strategy for mycoplasma infections thatcause infections such as pneumonia.

Homozygous non-human animals which were born at the expected Mendelianratios include a deficient type being deficient in a protein thatspecifically recognizes a mycoplasma-derived lipoprotein/lipopeptide andits wild-type littermate, and precise comparative experiments can beconducted at an individual level by using the deficient type ofhomozygous non-human animals and its wild-type littermatesimultaneously. Therefore, it is desirable to use a wild-type non-humananimal, preferably a wild-type non-human animal which is the samespecies as, more preferably the littermate of, a non-human animal whosefunction of a gene that encodes a protein that specifically recognizes amycoplasma-derived lipoprotein/lipopeptide is deficient on itschromosome, for instance, for the screening of an inhibitor or apromoter for a response to a mycoplasma-derived lipoprotein/lipopeptideof the present invention mentioned below.

The non-human animal model unresponsive to a mycoplasma-derivedlipoprotein/lipopeptide of the present invention and an immune cell suchas a macrophage, a splenocyte or a dendric cell derived from thenon-human animal model can be used for the elucidation of an actionmechanism of a mycoplasma-derived lipoprotein/lipopeptide, and also forscreenings of an inhibitor or a promoter for mycoplasma infections suchas mycoplasma pneumonia, or an inhibitor or a promoter for a response toa mycoplasma-derived lipoprotein/lipopeptide, such as an agonist or anantagonist of TLR6, etc. The method for screening the inhibitor or thepromoter for mycoplasma infections such as pneumonia, or the inhibitoror the promoter for a response to a mycplasma-derivedlipoprotein/lipopeptide, such as an agonist or an antagonist of TLR6, isexplained with examples below.

As the method for screening an inhibitor or a promoter for a response toa mycoplasma-derived lipoprotein/lipopeptide of the present invention, amethod wherein with the use of an immune cell such as a macrophage, asplenocyte or a dendric cell derived from a non-human animal modelunresponsive to a mycoplasma-derived lipoprotein/lipopeptide, a subjectmaterial and a mycoplasma-derived lipoprotein/lipopeptide, a response toa mycoplasma-derived lipoprotein/lipopeptide in the immune cell ismeasured/evaluated, and a method wherein with the use of a non-humananimal model unresponsive to a mycoplasma-derivedlipoprotein/lipopeptide, a subject material and a mycoplasma-derivedlipoprotein/lipopeptide, a response to a mycoplasma-derivedlipoprotein/lipopeptide in the non-human animal model ismeasured/evaluated, etc., are exemplified.

Examples of the above-mentioned method for screening with the use of animmune cell derived from a non-human animal model unresponsive to amycoplasma-derived lipoprotein/lipopeptide include a method comprisingthe steps of: contacting an immune cell obtained from a non-human animalmodel unresponsive to a mycoplasma-derived lipoprotein/lipopeptide witha subject material in vitro beforehand; culturing the immune cell in thepresence of a mycoplasma-derived lipoprotein/lipopeptide, andmeasuring/evaluating a response to a mycoplasma-derivedlipoprotein/lipopeptide in the immune cell, and a method comprising thesteps of: contacting an immune cell obtained from a non-human animalmodel unresponsive to a mycoplasma-derived lipoprotein/lipopeptide witha mycoplasma-derived lipoprotein/lipopeptide in vitro beforehand;culturing the immune cell in the presence of a subject material, andmeasuring/evaluating a response to a mycoplasma-derivedlipoprotein/lipopeptide in the immune cell.

In addition to the methods mentioned above, the following methods areexemplified: a method comprising the steps of: a subject material isadministered in advance to a non-human animal model unresponsive to amycoplasma-derived lipoprotein/lipopeptide; an immune cell obtained fromthe non-human animal is cultured in the presence of a mycoplasma-derivedlipoprotein/lipopeptide; a response to a mycoplasma-derivedlipoprotein/lipopeptide in the immune cell is measured/evaluated, and amethod comprising the steps of: a subject material is administered inadvance to a non-human animal model unresponsive to a mycoplasma-derivedlipoprotein/lipopeptide of the present invention; the non-human animalis made to be infected with a mycoplasma-derivedlipoprotein/lipopeptide; a response to a mycoplasma-derivedlipoprotein/lipopeptide in an immune cell obtained from the non-humananimal is measured/evaluated.

Further, a method comprising the steps of: a non-human animal modelunresponsive to a mycoplasma-derived lipoprotein/lipopeptide of thepresent invention is made to be infected with a mycoplasma-derivedlipoprotein/lipopeptide in advance; an immune cell obtained from thenon-human animal is cultured in the presence of a subject material, anda response to a mycoplasma-derived lipoprotein/lipopeptide in the immunecell is measured/evaluated, and a method comprising the steps of: anon-human animal model unresponsive to a mycoplasma-derivedlipoprotein/lipopeptide of the present invention is made to be infectedwith a mycoplasma-derived lipoprotein/lipopeptide in advance; a subjectmaterial is administered to the non-human animal, and a response to amycoplasma-derived lipoprotein/lipopeptide in an immune cell obtainedfrom the non-human animal is measured/evaluated are exemplified.

On the other hand, the following methods are exemplified as the methodwherein with the use of a non-human animal model unresponsive to amycoplasma-derived lipoprotein/lipopeptide of the present invention, asubject material and a mycoplasma-derived lipoprotein/lipopeptide, aresponse to a mycoplasma-derived lipoprotein/lipopeptide in thenon-human animal model is measured/evaluated: a method comprising thesteps of: a subject material is administered in advance to a non-humananimal model unresponsive to a mycoplasma-derivedlipoprotein/lipopeptide; the non-human animal model is made to beinfected with a mycoplasma-derived lipoprotein/lipopeptide; a responseto a mycoplasma-derived lipoprotein/lipopeptide in the non-human animalmodel is measured/evaluated, and a method comprising the steps of: anon-human animal model unresponsive to a mycoplasma-derivedlipoprotein/lipopeptide is made to be infected with a mycoplasma-derivedlipoprotein/lipopeptide in advance; a subject material is administeredto the non-human animal model, and a response to a mycoplasma-derivedlipoprotein/lipopeptide in the non-human animal model ismeasured/evaluated.

In the present invention, the measurement/evaluation of a response to amycoplasma-derived lipoprotein/lipopeptide means ameasurement/evaluation of a function to specifically react with amycoplasma-derived lipoprotein/lipopeptide to transmit signalsintracellularly. As such signal transmitting function, a function toproduce cytokines such as TNF-á, IL-6, IL-12 and IFN-ã, a function toproduce nitrite ion, a function to proliferate cells, a function toexpress antigens such as CD40, CD80, CD86 and MHC class II on thesurface of cells, and a function to activate molecules in the signalingpathway of TLR9, such as NF-êB, JNK and IRAK are specificallyexemplified, but not limited to these functions. Further, asaforementioned, in the measurement/evaluation of a response to amycoplasma-derived lipoprotein/lipopeptide, it is preferable to evaluatethe response in comparison to the measured value of a wild-typenon-human animal, a wild-type non-human animal littermate in particular,as a control, because there will be no dispersion caused by individualdifferences.

As it is revealed by the non-human animal model specifically deficientin reactivity to a mycoplasma-derived lipoprotein/lipopeptide of thepresent invention that TLR6 is specifically involved in the recognitionof a mycoplasma-derived lipoprotein/lipopeptide, these non-human animalmodels are presumed to serve as extremely useful model animals forprojecting a therapeutic strategy for mycoplasma infections such ashuman mycoplasma pneumonia which is a primary a typical pneumonia causedby M. pneumoniae, bovine pleuropneumonia caused by M. mycoides,ovine/caprine mastitis caused by M. agalacliae, or chicken respiratorydisease caused by M. gallisepticum. In addition, there is a chance thatan agonist or an antagonist of TLR6 is an inhibitor or a promoter forvarious kinds of mycoplasma infections mentioned above, and is a usefulsubstance for diagnosis/treatment of diseases caused by deficiency orabnormality in TLR6 activity, etc.

The present invention will be explained more specifically with examplesand a reference below, but the technical scope of the present inventionis not limited to these examples etc. Reference (Generation of TLR2knockout mouse) TLR2 gene was screened from 129/SvJ mouse gene library(Stratagene) with a probe derived from a mouse EST clone (accessionnumber D77677) which is similar to human TLR2 gene, and subcloned into apBluescript vector (Stratagene), then characterized by restrictionenzyme mapping and DNA sequencing. A targeting vector was constructed byreplacing a gene fragment at an exon region 1.3 kb containing acytoplasmic domain of a TLR2 gene with pMC1-neo (Stratagene) having PolyA signal. The targeting vector was flanked by a 4.8 kb 5′ gene fragmentand a 1.0 kb 3′ gene fragment and contained a HSV-tk cassette at the 5′terminal. The targeting vector was linearized with SalI andelectroporated into E14.1 embryonic stem cells (ES cells). ES cellsbeing resistant to G418 and ganciclovir, and containing mutant TLR2allele were screened from the electroporated ES cells, and the screenedES cells were microinjected into blastocysts of C57BL/6 mice toconstruct chimeric mice. By mating this male chimeric mouse and a femaleC57BL/6 mouse, TLR2 knockout mouse was constructed (Immunity 11,443-451, 1999).

EXAMPLE 1 Generation of TLR6 Knockout Mouse

TLR6 gene was screened from 129/SvJ mouse gene library (Stratagene) witha probe derived from a mouse TLR6 gene (accession number AB020808), andsubcloned into a pBluescript II SK (+) vector (Stratagene), thencharacterized by restriction enzyme mapping and DNA sequencing. Atargeting vector was constructed by replacing a genetic site (about 6kb) encoding an intracellular domain and a transmembrane domain of themouse TLR6 with a neomycin-resistant gene cassette (pMC1-neo;Stratagene), and introducing a herpes simplex virus thymidine kinase(HSV-tk) as a negative selective marker (FIG. 1). The targeting vectorwas linearized and electroporated into E14.1 embryonic stem cells (EScells). 126 clones being resistant to G418 and ganciclovir were selectedand three clones were screened by PCR and Southern blotting.

Three targeted ES clones containing mutant TLR6 allele weremicroinjected into blastocysts of C57BL/6 mice to generate a chimericmouse. By mating this male chimeric mouse and a female C57BL/6 mouse, aheterozygous F1 mouse was generated, and a homozygous mouse (TLR6knockout mouse: TLR6−/−) was obtained by intercrossing the heterozygousF1 mice. Confirmation of the homozygous mouse was conducted by digestingeach genomic DNA extracted from a mouse tail with EcoRI and performingSouthern blotting with a probe shown in FIG. 1 (FIG. 2). The TLR6knockout mouse (TLR6−/−) of the present invention could be generated atthe expected Mendelian ratio, and did not show any obvious abnormalityuntil it came to 25 weeks old.

EXAMPLE 2 Preparation of Peritoneal Macrophage

Each of wild-type (wild-type), TLR6 knockout (TLR6−/−) and TLR2 knockout(TLR2−/−) mice were intraperitoneally injected with 2 ml of 4%thioglycollate medium (DIFCO). Three days later, peritoneal exudatecells were isolated from the peritoneal cavity of each mouse. Thesecells were cultured in RPMI1640 medium (GIBCO) supplemented with 10%fetal bovine serum (GIBCO) for 2 hours at 37.degree. C. and washed withice-cold Hank's buffered salt solution (HBSS; GIBCO) to removenonadherent cells. Adherent cells were used as peritoneal macrophagesfor the following experiments.

EXAMPLE 3 Responsiveness of Macrophages Derived from TLR6 Knockout Mouseto LPS, Lipoprotein Derived from Spirochaeta and Peptidoglycan Derivedfrom Staphylococcus aureus

The present inventors have already revealed that TLR2 is indispensablefor recognizing lipoprotein and peptidoglycan derived from bacteria andthat TLR4 is indispensable for recognizing LPS and lipoteichoic acid bygenerating TLR2 and TLR4 knockout mice. Therefore, responsiveness ofTLR6 knockout mouse to these LPS, lipoprotein derived from Spirochaeta,and peptidoglycan derived from Staphylococcus aureus was examined.Peritoneal macrophages (5×10⁴ cells) of wild-type and TLR6 knockout miceprepared in Example 2 were cultured for 24 hours together with 10 μM B.burgdorferi-derived lipoprotein OspA or OspC, 10 μM T. pallidum-derivedlipoprotein 17-kDa (17) or 47-kDa (47), 100 ng/ml LPS, or variousconcentrations of peptidoglycan (PGN) shown in FIG. 3. When culture wascompleted, concentrations of TNFá in culture supernatants were measuredby ELISA respectively. The results are shown in FIG. 3. These resultshave indicated that macrophages derived from a TLR6 knockout mouse(TLR6−/−) produce approximately the same amount of TNFá as macrophagesderived from a wild-type mouse in response to OspA, OspC, 17, 47, LPS,and PGN.

EXAMPLE 4 Responsiveness of Macrophages Derived from TLR6 Knockout Mouseto Lipopeptide

Responsiveness of peritoneal macrophages derived from a wild-type mouseor a TLR6 knockout mouse to lipopeptide was examined with synthetic Gramnegative bacteria-derived lipopeptide JBT3002 and syntheticmycoplasma-derived lipopeptide MALP-2. Peritoneal macrophages (5×10⁴cells) of wild-type and TLR6 knockout mice prepared in Example 2 werecultured for 24 hours together with various concentrations of JBT3002(provided by Dr. Z. Dong) or MALP-2 (provided by Dr. P. F. Muhlradt)shown in FIG. 4, and stimulated. When culture was completed, theproduction amounts of TNF-á (TNFá) and NO₂ ⁻ (NO) in culturesupernatants were measured (FIG. 4). NO₂ ⁻ was measured by the Greissmethod using NO₂/NO₃ Assay Kit (Dojindo Laboratories).

The above-mentioned results indicate that: peritoneal macrophagesderived from a wild-type mouse (wild-type) increased the productionamounts of TNF-á (TNFá) and NO₂ ⁻ (NO) in a manner dependent on doses ofGram negative bacteria-derived lipopeptide JBT3002 andmycoplasma-derived lipopeptide MALP-2, while peritoneal macrophagesderived from a TLR6 knockout mouse (TLR6−/−) increased the productionamounts of TNF-á and NO₂ ⁻ in a manner dependent on dose of Gramnegative bacteria-derived lipopeptide JBT3002 as in the case ofperitoneal macrophages derived from a wild-type mouse, but produced noTNF-á and NO₂ ⁻ in response to any concentrations of mycoplasma-derivedlipopeptide MALP-2 (FIG. 4). From these results, it is revealed that amycoplasma-derived lipoprotein/lipopeptide activates macrophages viaTLR6.

EXAMPLE 5 Activation of Intracellular Signaling Pathway via TLR6

It is known that TLR signals activate IRAK, which is serine/threoninekinase, via an adapter molecule MyD88 and then activate MAP kinase andNF-êB (Immunity 11, 115-122, 1999). In addition, it is also known that amycoplasma-derived lipoprotein/lipopeptide causes vital reaction viaTLR2 and MyD88 signaling pathways (J. Immunol. 164, 554-557, 2000).Consequently, with wild-type, TLR6 knockout and TLR2 knockout mice,whether a bacteria-derived lipoprotein/lipopeptide activatesintracellular signaling molecules was examined. Peritoneal macrophages(1×10⁶ cells) of wild-type (wild-type) and TLR6 knockout (TLR6−/−) andTLR2 knockout (TLR2−/−) mice prepared in Example 2 were stimulated with0.1 ng/ml JBT3002 or 0.3 ng/ml MALP-2 for 20 or 40 minutes, andnucleoproteins were extracted from macrophages of each mouse andincubated with a specific probe containing DNA binding site of NF-êB,then subjected to electrophoresis and visualized by autoradiography(FIG. 5).

With the result that the activation of NF-êB in response to thestimulation with MALP-2 or JBT3002 was observed in macrophages derivedfrom a wild-type mouse. In macrophages derived from a TLR6 knockoutmouse, though no activation in response to MALP-2 was observed,activation in response to JBT3002 was observed. Further, in macrophagesderived from a TLR2 knockout mouse, no activation was observed inresponse to MALP-2 and JBT3002. These findings have revealed that TLR6is specifically involved in the recognition of a mycoplasma-derivedlipoprotein/lipopeptide. In addition, it is presumed that both TLR6 andTLR2 are indispensable for recognizing a mycoplasma-derivedlipoprotein/lipopeptide, and that these two molecules form a heterodimerto transmit mycoplasma-derived lipoprotein/lipopeptide signals.

INDUSTRIAL APPLICABILITY

The non-human animal model unresponsive to a mycoplasma-derivedlipoprotein/lipopeptide of the present invention such as a TLR6 knockoutmouse is unresponsive only to a mycoplasma-derivedlipoprotein/lipopeptide. Therefore, the use of this non-human animalmodel makes it possible to screen an inhibitor or a promoter formycoplasma infections such as mycoplasma pneumonia, or an inhibitor or apromoter for responsiveness to a mycoplasma-derivedlipoprotein/lipopeptide such as an agonist or an antagonist of TLR6, andmoreover, to obtain novel information useful for elucidating a molecularmechanism of the occurrence of infections caused by bacteria includingMycoplasma.

1. A transgenic mouse whose genome comprises a homozygous disruption ofthe TLR-6 gene, said TLR-6 gene encoding a polypeptide that recognizes aMALP-2 mycoplasma-derived lipoprotein/lipopeptide, wherein macrophagecells isolated from the transgenic mouse are unresponsive to MALP-2. 2.A macrophage cell isolated from a transgenic mouse whose genomecomprises a homozygous disruption of the TLR-6 gene, said TLR-6 geneencoding a polypeptide that recognizes a MALP-2 mycoplasma-derivedlipoprotein/lipopeptide, wherein the macrophage cell is unresponsive toMALP-2.